The interactome of the N-terminus of band 3 regulates red blood cell metabolism and storage quality

Abstract

Band 3 (anion exchanger 1 - AE1) is the most abundant membrane protein in red blood cells (RBCs), the most abundant cell in the human body. A compelling model, based on indirect evidence, posits that - at high oxygen saturation - the N-term cytosolic domain of AE1 binds to and inhibits glycolytic enzymes, thus diverting metabolic fluxes to the pentose phosphate pathway to generate reducing equivalents. Dysfunction of this mechanism occurs during RBC aging or storage under blood bank conditions, suggesting a role for AE1 in the regulation of blood storage quality and efficacy of transfusion – a life-saving intervention for millions of recipients worldwide. Here we leverage two murine models carrying genetic ablations of AE1 to provide the first direct mechanistic evidence of its role in metabolic regulation and blood storage quality. Observations in mice phenocopied those in a human subject lacking expression of AE11-11 (band 3 Neapolis), while common polymorphisms in the region coding for AE11-56 increased susceptibility to osmotic hemolysis in healthy blood donors. Through thermal proteome profiling and cross-linking proteomics, we provide the first comprehensive analysis of the RBC interactome, with a focus on AE11-56 and validate recombinant AE1 interactions with glyceraldehyde 3-phosphate dehydrogenase (GAPDH). Finally, we show that incubation with a cell-penetrating AE11-56 peptide can rescue the metabolic defect in glutathione recycling and boost post-transfusion recoveries of stored RBCs from healthy human donors and genetically ablated mice, paving the way for the in vivo metabolic manipulation of RBCs facing oxidant stress – a landmark of many diseases.Key pointsGenetic ablation of N-term of band 3 results in significant metabolic aberrations and poor post-transfusion recoveries in mice and humans;Structural studies on the N-term of band 3 reveal a complex interactome with several enzymes, including GAPDH;

Circulating primitive murine erythroblasts undergo complex proteomic and metabolomic changes during terminal maturation

“…28 It remains to be assessed whether rare genetic anomalies impacting the N-terminus of band 3 also result in impaired primitive erythropoiesis, on top of well-established defects in mature red blood cell redox homeostasis, e.g., band 3 Neapolis is accompanied by reticulocytosis and erythrocytes with a compromised structure and metabolism. 62,63 Since this mechanism can also be regulated by phosphorylation of the Nterminus of band 3 by Lyn and Syk kinase, it will be interesting to probe whether the activity of these enzymes plays a role in primitive erythroid maturation by regulating the band 3 respiratory metabolon, 29,64 and overall interactome 65 , on top of their well-established role in definitive erythroid expansion, maturation and differentiation via phosphorylation of GAB2/Akt/FoxO3 and STAT5. 66,67 Significant depletion of mitochondrial proteins, including structural components, proteins involved in mitochondrial RNA metabolism, electron transport chain components and TCA cycle enzymes, was accompanied by a significant depression of steady state levels of carboxylic acids, amino acids, acylcarnitines and free fatty acids.…”

Cross-talk between red blood cells and plasma influences blood flow and omics phenotypes in severe COVID-19

“…Proteomics analyses are performed via filter aided sample preparation (FASP) digestion and nano UHPLC-MS/MS identification (TIMS TOF Pro 2 Single Cell Proteomics, Bruker Daltonics, Bremen, Germany), as previously described (24).…”

Mouse background genetics in biomedical research: The devil's in the details

“…The N-terminus of Band3 allows RBCs to modify their metabolism and function in response to the oxygen saturation of hemoglobin. [39][40][41] We acquired three strains of previously reported mice in which the Nterminus of the endogenous murine Band3 was replaced with either the canonical or mutated N-terminus of human Band3. All mice were reported as having been backcrossed to a B6 background.…”

“…Band3 is the most abundant membrane protein of red blood cells (RBCs). The N‐terminus of Band3 allows RBCs to modify their metabolism and function in response to the oxygen saturation of hemoglobin 39–41 . We acquired three strains of previously reported mice in which the N‐terminus of the endogenous murine Band3 was replaced with either the canonical or mutated N‐terminus of human Band3.…”